Microfluidized oil-in-water emulsions and vaccine compositions

ABSTRACT

This invention provides submicron oil-in-water emulsions useful as a vaccine adjuvant for enhancing the immunogenicity of antigens. The present invention also provides vaccine compositions containing an antigen combined with such emulsions intrinsically or extrinsically. Methods of preparing the emulsions and vaccines are also provided by the present invention.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/460,301, filed on Apr. 4, 2003.

FIELD OF INVENTION

[0002] This invention relates generally to the field of vaccines andparticularly, to adjuvant formulations for enhancing immune response inveterinary animals. In particular, the invention relates to the use of asubmicron oil-in-water emulsion as a vaccine adjuvant for enhancing theimmunogenicity of antigens. Submicron oil-in-water emulsionformulations, vaccine compositions containing an antigen incorporatedinto such emulsions, as well as methods of preparing the emulsions andvaccines, are provided by the present invention.

BACKGROUND OF THE INVENTION

[0003] Bacterial, viral, parasitic and mycoplasma infections are widespread in the veterinary animals such as cattle, swine and companionanimal. Diseases caused by these infectious agents are often resistantto antimicrobial pharmaceutical therapy, leaving no effective means oftreatment. Consequently, a vaccinology approach is increasingly used tocontrol the infectious disease in the veterinary animals. A wholeinfectious pathogen can be made suitable for use in a vaccineformulation after chemical inactivation or appropriate geneticmanipulation. Alternatively, a protein subunit of the pathogen can beexpressed in a recombinant expression system and purified for use in avaccine formulation.

[0004] Adjuvant generally refers to any material that increases thehumoral and/or cellular immune response to an antigen. The traditionalvaccines are composed of crude preparation of killed pathogenicmicroorganisms, and the impurities associated with the cultures ofpathological microorganisms could act as adjuvant to enhance the immuneresponse. However, when homogeneous preparations of pathologicalmicroorganisms or purified protein subunits are used as antigens forvaccination, the immunity invoked by such antigens is poor and theaddition of certain exogenous materials as adjvuant therefore becomesnecessary. Further, synthetic and subunit vaccines are expensive toproduce. Therefore, with the aid of adjuvant, a smaller dose of antigenmay be required to stimulate the immune response, thereby saving theproduction cost of vaccines.

[0005] Adjuvants are known to act in a number of different ways toenhance the immune response. Many adjuvants modify the cytokine networkassociated with immune response. These immunomodulatory adjuvants canexert their effect even when they are not together with antigens. Ingeneral the immunomodulatory adjuvants cause a general up-regulation ofcertain cytokines and a concomitant down regulation of others leading toa cellular Th1 and/or a humoral Th2 response.

[0006] Some adjuvants have the ability to preserve the conformationalintegrity of an antigen so that the antigens can be efficientlypresented to appropriate immune effector cells. As a result of thispreservation of antigen conformation by the adjuvant formulation, thevaccine would have an increased shelf-life such as that shown for immunestimulating complexes (ISCOMs). Ozel M.,et.al.; Quarternary Structure ofthe Immunestimmulating Complex (Iscom), J.of Ultrastruc. and Molec.Struc. Res. 102, 240-248 (1989).

[0007] Some adjuvants have the property of retaining the antigen as adepot at the site of injection. As a result of this depot effect theantigen is not quickly lost by liver clearance. Aluminum salts and thewater-in-oil emulsions act through this depot effect for a shorterduration. For example, one can obtain a long-term depot by usingFreund's complete adjuvant (FCA) which is an water-in-oil emulsion. FCAtypically remains at the injection site until biodegradation permitsremoval of the antigen by antigen-presenting cells.

[0008] Based on their physical nature, adjuvants can be grouped undertwo very broad categories, namely particulate adjvuants andnon-particulate adjvuants. Particulate adjuvants exist asmicroparticles. The immunogen is either able to incorporate or associatewith the microparticles. Aluminum salts, water-in-oil emulsions,oil-in-water emulsions, immune stimulating complexes, liposomes, andnano- and microparticles are examples of particulate adjuvants. Thenon-particulate adjuvants are generally immunomodulators and they aregenerally used in conjunction with particulate adjuvants. Muramyldipeptide (an adjuvant-active component of a peptidoglycan extractedfrom Mycobacteria), non-ionic block copolymers, Saponins (a complexmixture of triterpenoids extracted from the bark of the Quillajasaponaria tree), Lipid A (a disaccharide of glucosamine with twophosphate groups and five or six fatty acid chains generally C12 to C16in length), cytokines, carbohydrate polymers, derivatizedpolysaccharides, and bacterial toxins such as cholera toxin and E. colilabile toxin (LT) are examples of non-particulate adjuvants.

[0009] Some of the best-known adjuvants are combination ofnon-particulate immunomodulators and particulate materials which couldimpart depot effect to the adjuvant formulation. For example, FCAcombines the immunomodualtory properties of Mycobacterium tuberculosiscomponents along with the short-term depot effect of oil emulsions.

[0010] Oil emulsions have been used as vaccine adjuvant for a long time.Le Moignic and Pinoy found in 1916 that a suspension of killedSalmonella typhimurium in mineral oil increased the immune response.Subsequently in 1925, Ramon described starch oil as one of thesubstances augmenting the antitoxic response to diptheria toxoid.However, the oil emulsions did not become popular until 1937 when Freundcame out with his adjuvant formulation now known as Freund's CompleteAdjuvant (FCA). FCA is a water-in-oil emulsion composed of mineral(paraffin) oil mixed with killed Mycobateria and Arlacel A. Arlacel A isprincipally mannide monooleate and is used as an emulsifying agent.Although FCA is excellent in inducing an antibody response, it causessevere pain, abscess formation, fever and granulomatous inflammation. Toavoid these undesirable side reactions, Incomplete Freund's Adjuvant(IFA) was developed. IFA is similar to FCA in its composition except forthe absence of mycobacterial components. IFA acts through depotformulation at the site of injection and slow release of the antigenwith stimulation of antibody-producing cells.

[0011] Another approach to improve FCA was based on the notion thatreplacing the mineral oil with a biocompatible oil would help eliminatethe reactions associated with FCA at the injection site. It was alsobelieved that the emulsion should be oil-in-water rather thanwater-in-oil, because the latter produces a long-lasting depot at theinjection site. Hilleman et al. described an oil-based adjuvant“Adjuvant 65”, consisting of 86% peanut oil, 10% Arlacel A as emulsifierand 4% aluminum monostearate as stabilizer. Hilleman, 1966, Prog. Med.Virol. 8:131-182; Hilleman and Beale, 1983, in New Approaches to VaccineDevelopment (Eds. Bell, R. and Torrigiani, G.), Schwabe, Basel. Inhumans, Adjuvant 65 was safe and potent but exhibited less adjuvanticitythan IFA. Nevertheless, the use of Adjvuant 65 was discontinued due toreactogenicity for man with certain lots of vaccine and reduction inadjuvanticity when a purified or synthetic emulsifier was used in placeof Arlacel A. U.S. Pat. Nos. 5,718,904 and 5,690,942 teach that themineral oil in the oil-in-water emulsion can be replaced withmetabolizable oil for the purpose of improving the safety profile.

[0012] Besides the adjuvanticity and safety, the physical appearance ofan emulsion is also an important commercial consideration. Physicalappearance depends on the stability of the emulsion. Creaming,sedimentation and coalescence are indicators of the emulsioninstability. Creaming occurs when oil and aqueous phases of the emulsionhave different specific gravity. Creaming also occurs when the initialdroplet size of the emulsion is large and the emulsion droplets are nothaving any Brownian motion. When the droplet size is large, there is atendency for the interfacial rupture and the droplets coalesce intolarge particles. The stability of the emulsion is determined by a numberof factors such as the nature and amount of emulsifier used, the size ofthe droplet size in the emulsion, and the difference in the densitybetween the oil and water phase.

[0013] Emulsifiers promote stabilization of dispersed droplet byreducing the interfacial free energy and creating physical orelectrostatic barriers to droplet coalescence. Nonionic as well as ionicdetergents have been used as emulsifiers. Nonionic emulsifiers orient atthe interface and produce relatively bulky structures, which leads tosteric avoidance of the dispersed droplets. Anionic or cationicemulsifiers induce formation of an electrical double layer by attractingcounter ions; the double layer repulsive forces cause droplets to repelone another when they approach.

[0014] Besides using the emulsifiers, the stability of the emulsion canalso be achieved through reducing the droplet size of the emulsion bymechanical means. Typically propeller mixers, turbine rotors, colloidmills, homogenizers, and sonicators have been used to manufactureemulsions. Microfluidization is another way to increase the homogeneityof the droplet size in the emulsion. Microfluidization can produce anelegant, physically stable emulsion with consistent particle size in thesubmicron range. Besides increasing the stability of the emulsion, theprocess of microfluidization allows terminal filtration which is apreferred way of ensuring the sterility of the final product. Moreover,submicron oil particles can pass from injection sites into thelymphatics and then to lymph nodes of the drainage chain, blood andspleen. This reduces the likelihood of establishing an oily depot at theinjection site which may produce local inflammation and significantinjection site reaction.

[0015] Microfluidizers are now commercially available. Emulsionformation occurs in a microfluidizer as two fluidized streams interactat high velocities within an interaction chamber. The microfluidizer isair or nitrogen driven and can operate at internal pressures in theexcess of 20,000 psi. U.S. Pat. No. 4,908,154 teaches the use ofmicrofluidizer for obtaining emulsions essentially free of anyemulsifying agents.

[0016] A number of submicron oil-in-water adjuvant formulations havebeen described in the literature. U.S. Pat. No. 5,376,369 teaches asubmicron oil-in-water emulsion adjuvant formulation known as SyntaxAdjuvant Formulation (SAF). SAF contains squalene or squalane as the oilcomponent, an emulsion-forming amount of Pluronic L121(polyoxy-proplyene-polyoxyethylene) block polymer and animmunopotentiating amount of muramyldipeptide. Squalene is a linearhydrocarbon precursor of cholesterol found in many tissues, notably inthe livers of sharks and other fishes. Squalane is prepared byhydrogenation of squalene and is fully saturated. Both squalene andsqualane can be metabolized and have a good record of toxicologicalstudies. Squalene or squalane emulsions have been used in human cancervaccines with mild side effects and a desirable efficacy. See, e.g.,Anthony C. Allison, 1999, Squalene and Squalane emulsions as adjuvants,Methods 19:87-93.

[0017] U.S. Pat. No. 6,299,884 and International Patent Publication WO90/14837 teach that the polyoxy-proplyene-polyoxyethylene blockcopolymers are not essential for the formation of submicron oil-in-wateremulsion. Moreover, these references teach the use of non-toxic,metabolizable oil and expressly exclude the use of mineral oil and toxicpetroleum distillate oils in their emulsion formulations.

[0018] U.S. Pat. No. 5,961,970 teaches yet another submicronoil-in-water emulsion to be used as a vaccine adjuvant. In the emulsiondescribed in this patent, the hydrophobic component is selected from thegroup consisting of a medium chain triglyceride oil, a vegetable oil anda mixture thereof. The surfactant included in this emulsion can be anatural biologically compatible surfactant such as phospholipid (e.g.,lecithin) or a pharmaceutically acceptable non-natural surfactant suchas TWEEN-80. This patent also teaches incorporating the antigen into theemulsion at the time the emulsion is formed, in contrast to mixing theantigen with the emulsion after the emulsion has been independently andextrinsically formed.

[0019] U.S. Pat. No. 5,084,269 teaches that an adjuvant formulationcontaining lecithin in combination with mineral oil causes a decrease inirritation within the host animal and simultaneously induces increasedsystemic immunity. The adjuvant formulation resulting from U.S. Pat.5,084,269 is commercially used in veterinary vaccines under the tradename AMPHIGEN®. The AMPHIGEN® formulation is made up of micelles—oildroplets surrounded by lecithin. These micelles allow more whole cellantigens to attach than traditional oil-based adjuvants. Moreover, theAMPHIGEN®-based vaccine formulations contain a low oil content of 2.5 to5% mineral oil, compared to other vaccine formulations containing oiladjuvants, which typically contain from 10% to 20% oil. Its low oilcontent makes this adjuvant-based vaccine formulation less irritating totissues at the injection site, resulting in fewer lesions and less trimat slaughter. In addition, the lecithin coating surrounding the oildroplets further reduces injection site reactions resulting in a vaccinethat is both safe and efficacious.

[0020] The AMPHIGEN® formulation is used as an adjuvant in a number ofveterinary vaccines and there is need to maintain the physicalappearance of the vaccine product during short and long storage periodsas well as at the time of reconstitution. In addition, a lyophilizedantigen is mixed with the pre-made adjuvant formulation just before theinjection. This practice does not always ensure that there is a uniformdistribution of the antigen within the oil-in-water emulsion and theappearance of the emulsion may not be desirable. Moreover, uponstanding, the homogenized emulsion can show phase separation. Therefore,there exists a need for a stable adjuvant formulation which does notshow phase separation upon long shelf-life. One way to prevent the phaseseparation is to reduce the droplet size and increase the particlehomogeneity of the emulsion. While the process of microfluidization ofmetabolizable oil-based emulsion formulations has been documented,microfluidization of oil-in-water emulsions such as the AMPHIGEN®formulation has not yet been carried out.

[0021] In the present invention, microfluidization has been used tobring the size of lecithin-surrounded mineral oil droplets to submicronsize. Unexpectedly, it has been discovered by the present inventors thatmicrofluidization of vaccine formulations adjuvanted with anoil-in-water emulsion comprised of a mixture of lecithin and oil notonly improves the physical appearance of the formulations, but alsoenhances the immunizing effects of the formulations. Microfluidizedformulations are also characterized by an improved safety profile.

SUMMARY OF THE INVENTION

[0022] It has been unexpectedly discovered by the present inventors thatthe adjuvant activity and the safety profile of non-metabolizable oilbased oil-in-water emulsions can be improved through microfluidization.Antigens incorporated in microfluidized emulsions are stable even whenthe antigens are intrinsically incorporated into the emulsions prior tomicrofluidization.

[0023] Accordingly, in one embodiment, the present invention providessubmicron oil-in-water emulsion formulations useful as a vaccineadjuvant. The submicron oil-in-water emulsions of the present inventionare composed of a non-metabolizable oil, at least one surfactant, and anaqueous component, where the oil is dispersed in the aqueous componentwith an average oil droplet size in the submicron range. A preferrednon-metabolizable oil is light mineral oil. Preferred surfactantsinclude lecithin, TWEEN®-80 and SPAN®-80.

[0024] A preferred oil-in-water emulsion provided by the presentinvention is composed of an AMPHIGEN®formulation.

[0025] The oil-in-water emulsions of the present invention can includeadditional components that are appropriate and desirable, includingpreservatives, osmotic agents, bioadhesive molecules, andimmunostimulatory molecules. Preferred immunostimulatory moleculesinclude, e.g., Quil A, cholesterol, GPI-0100,dimethyldioctadecylammonium bromide (DDA).

[0026] In another embodiment, the present invention provides methods ofpreparing a submicron oil-in-water emulsion. According to the presentinvention, the various components of the emulsion, including oil, one ormore surfactants, an aqueous component and any other componentappropriate for use in the emulsion, are mixed together. The mixture issubjected to a primary emulsification process to form an oil-in-wateremulsion, which is then passed through a microfluidizer to obtain anoil-in-water emulsion with droplets of less than 1 micron in diameter,preferably with a mean droplet size of less than 0.5 micron.

[0027] In still another embodiment, the present invention providesvaccine compositions which contain an antigen and a submicronoil-in-water emulsion described hereinabove. The antigen is incorporatedinto the emulsion either extrinsically or intrinsically, preferably,intrinsically.

[0028] The antigen which can be included in the vaccine compositions ofthe present invention can be a bacterial, fungal, or viral antigen, or acombination thereof. The antigen can take the form of an inactivatedwhole or partial cell or virus preparation, or the form of antigenicmolecules obtained by conventional protein purification, geneticengineering techniques or chemical synthesis.

[0029] In a further embodiment, the present invention provides methodsof preparing vaccine compositions containing an antigen or antigenscombined with a submicron oil-in-water emulsion.

[0030] In preparing the vaccine compositions of the present invention,the antigen(s) can be combined either intrinsically (e.g., prior tomicrofluidization) or extrinsically (e.g., after microfluidization) withthe components of the oil-in-water emulsion. Preferably, the antigen iscombined with the components of the oil-in-water emulsion intrinsically.

[0031] In still another embodiment, the present invention providesvaccine compositions which contain a microencapsulated antigen and asubmicron oil-in-water emulsion described hereinabove, where themicroencapsulated antigen is combined with the emulsion extrinsically.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 depicts the process for the batch preparation ofnon-microfluidized vaccine compositions. In this process the variousvaccine components are added to the addition vessel on the left andultimately pumped into the blend vessel where the components are mixedtogether through simple mechanical means.

[0033]FIG. 2 depicts the process for preparation of microfluidizedvaccine compositions containing intrinsically incorporated antigen. Thevarious vaccine components are added to the addition vessel andtransferred to the pre-emulsion blending unit for mixing through simplemechanical means. Subsequently, the emulsion is passed through amicrofluidizer and is collected in the post-microfluidization chamber.

[0034]FIG. 3 depicts the droplet size distribution of thenon-microfluidized AMPHIGEN® formulation-based vaccine, themicrofluidized AMPHIGEN® formulation-based vaccine, and the bench blendvaccine preparation.

[0035]FIG. 4 shows absence of phase separation in the microfluidizedvaccine preparation.

[0036]FIG. 5 depicts a comparison of the stability of antigensintrinsically incorporated in microfluidized AMPHIGEN® formulation-basedvaccine preparation (A907505) and three control, non-microfluidizedAMPHIGEN®) formulation-based vaccine preparations (A904369, A904370, andA904371). All four vaccine preparations were stored at 4° C. for twoyears. At different points during the storage (0, 6, 12 or 24 months),all four formulations were used to vaccinate the three months old cows.Vaccination was done Day 0 and 21 with a 2 ml vaccine dose and the serawere collected two weeks post second vaccination. Neutralizing antibodytiter for BVD Type II virus was determined in each of the serum samples.The data are presented as the geometric mean for 5 animals.

[0037]FIG. 6 shows least squares mean rectal temperature of cattle priorto and following administration of microfluidized and non-microfluidizedvaccines. T01: Placebo group—single dose; T02: Placebo group—Doubledose; T03: Non-microfluidized formulation—Single Dose; T04:Non-microfluidized formulation—Double dose; T05: Microfluidizedformulation—Single Dose; T06: Microfluidized formulation—Double dose.

[0038]FIG. 7 depicts least squares mean injection site reaction volumesobserved in cattle following administration of non-microfluidized andmicrofluidized vaccine formulations. T03: Non-microfluidizedformulation—Single Dose; T04: Non-microfluidized formulation—Doubledose; T05: Microfluidized formulation—Single Dose; T06: Microfluidizedformulation—Double dose.

[0039]FIG. 8 depicts geometric mean IgG titers for recombinant PauAantigen from Streptococcus uberis after vaccination with the variousvaccine formulations containing both recombinant PauA antigen and E.coli whole cell antigen.

[0040]FIG. 9 depicts geometric mean IgG titers for E. coli whole cellantigen from Streptococcus uberis after vaccination with the variousvaccine formulations containing both recombinant PauA antigen and E.coli whole cell antigen.

[0041]FIGS. 10A and 10B depict the particle size distribution of aMicrofluidized Amphigen formulation at initial production (FIG. 10A) andat 22 months post production (FIG. 10B).

DETAILED DESCRIPTION OF THE INVENTION

[0042] It has been unexpectedly discovered by the present inventors thatmicrofluidization of vaccine formulations adjuvanted with anoil-in-water emulsion comprised of a mixture of lecithin and mineral oilnot only improves the physical appearance of the vaccine formulations,but also enhances the immunizing effects of the vaccine formulations.Microfluidized vaccine formulations are also characterized by animproved safety profile.

[0043] Based on these discoveries, the present invention providessubmicron oil-in-water emulsions useful as an adjuvant in vaccinecompositions. Methods of making these submicron oil-in-water emulsionsby using a microfluidizer are also provided. Furthermore, the presentinvention provides submicron vaccine compositions in which an antigen iscombined with a submicron oil-in-water emulsion. Methods for making suchvaccine compositions are also provided. The present invention furtherprovides vaccine compositions containing microencapsulated antigenscombined with a submicron oil-in-water emulsion and methods for makingsuch vaccines.

[0044] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the followingsubsections which describe or illustrate certain features, embodimentsor applications of the invention.

[0045] Submicron Oil-In-Water Emulsions

[0046] In one embodiment, the present invention provides submicronoil-in-water emulsion formulations useful as a vaccine adjuvant. Thesubmicron oil-in-water emulsions of the present invention enhance theimmunogenicity of antigens in vaccine compositions, are safe foradministration to animals and stable during storage.

[0047] The submicron oil-in-water emulsions of the present invention arecomposed of a non-metabolizable oil, at least one surfactant, and anaqueous component, where the oil is dispersed in the aqueous componentwith an average oil droplet size in the submicron range.

[0048] By “submicron” is meant that the droplets are of a size of lessthan 1 μm (micron) and the average or mean oil droplet size is less than1 μm. Preferably, the mean droplet size of the emulsion is less than 0.8μm; more preferably, less than 0.5 μm; and even more preferably, lessthan 0.4 μm, or about 0.1-0.3 μm.

[0049] The “mean droplet size” is defined as the Volume Mean Diameter(VMD) particle size within a volume distribution of particle sizes. TheVMD is calculated by multiplying each particle diameter by the volume ofall particles of that size and summing. This is then divided by thetotal volume of all particles.

[0050] The term “non-metabolizable oil” as used herein refers to oilsthat cannot be metabolized by the body of the animal subject to whichthe emulsion is administered.

[0051] The terms “animal” and “animal subject” as used herein refer toall non-human animals, including cattle, sheep, and pigs, for example.

[0052] Non-metabolizable oils suitable for use in the emulsions of thepresent invention include alkanes, alkenes, alkynes, and theircorresponding acids and alcohols, the ethers and esters thereof, andmixtures thereof. Preferably, the individual compounds of the oil arelight hydrocarbon compounds, i.e., such components have 6 to 30 carbonatoms. The oil can be synthetically prepared or purified from petroleumproducts. Preferred non-metabolizable oils for use in the emulsions ofthe present invention include mineral oil, paraffin oil, andcycloparaffins, for example.

[0053] The term “mineral oil” refers to a mixture of liquid hydrocarbonsobtained from petrolatum via a distillation technique. The term issynonymous with “liquefied paraffin”, “liquid petrolatum” and “whitemineral oil.” The term is also intended to include “light mineral oil,”i.e., oil which is similarly obtained by distillation of petrolatum, butwhich has a slightly lower specific gravity than white mineral oil. See,e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.:Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil canbe obtained from various commercial sources, for example, J. T. Baker(Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferredmineral oil is light mineral oil commercially available under the nameDRAKEOL®.

[0054] Typically, the oil component of the submicron emulsions of thepresent invention is present in an amount from 1% to 50% by volume;preferably, in an amount of 10% to 45; more preferably, in an amountfrom 20% to 40%.

[0055] The oil-in-water emulsions of the present invention typicallyinclude at least one (i.e., one or more) surfactant. Surfactants andemulsifiers, which terms are used interchangeably herein, are agentswhich stabilize the surface of the oil droplets and maintain the oildroplets within the desired size.

[0056] Surfactants suitable for use in the present emulsions includenatural biologically compatible surfactants and non-natural syntheticsurfactants. Biologically compatible surfactants include phospholipidcompounds or a mixture of phospholipids. Preferred phospholipids arephosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithincan be obtained as a mixture of phosphatides and triglycerides bywater-washing crude vegetable oils, and separating and drying theresulting hydrated gums. A refined product can be obtained byfractionating the mixture for acetone insoluble phospholipids andglycolipids remaining after removal of the triglycerides and vegetableoil by acetone washing. Alternatively, lecithin can be obtained fromvarious commercial sources. Other suitable phospholipids includephosphatidylglycerol, phosphatidylinositol, phosphatidylserine,phosphatidic acid, cardiolipin, and phosphatidylethanolamine. Thephospholipids may be isolated from natural sources or conventionallysynthesized.

[0057] Non-natural, synthetic surfactants suitable for use in thesubmicron emulsions of the present invention include sorbitan-basednon-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants(commercially available under the name SPAN® or ARLACEL®), fatty acidesters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol estersof fatty acids from sources such as castor oil (EMULFOR);polyethoxylated fatty acid (e.g., stearic acid available under the nameSIMULSOL M-53), polyethoxylated isooctylphenol/formaldehyde polymer(TYLOXAPOL), polyoxyethylene fatty alcohol ethers (BRIJ®);polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethyleneisooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants arethe surfactants available under the name SPAN® and TWEEN®.

[0058] Preferred surfactants for use in the oil-in-water emulsions ofthe present invention include lecithin, Tween-80 and SPAN-80.

[0059] Generally speaking, the surfactant, or the combination ofsurfactants, if two or more surfactants are used, is present in theemulsion in an amount of 0.01% to 10% by volume, preferably, 0.1% to6.0%, more preferably 0.2% to 5.0%.

[0060] The aqueous component constitutes the continuous phase of theemulsion and can be water, buffered-saline or any other suitable aqueoussolution.

[0061] The oil-in-water emulsions of the present invention can includeadditional components that are appropriate and desirable, includingpreservatives, osmotic agents, bioadhesive molecules, andimmunostimulatory molecules.

[0062] It is believed that bioadhesive molecules can enhance thedelivery and attachment of antigens on or through the target mucoussurface conferring mucosal immunity. Examples of suitable bioadhesivemolecules include acidic non-naturally occurring polymers such aspolyacrylic acid and polymethacrylic acid (e.g., CARBOPOL®), CARBOMER);acidic synthetically modified natural polymers such ascarboxymethylcellulose; neutral synthetically modified natural polymerssuch as (hydroxypropyl) methylcellulose; basic amine-bearing polymerssuch as chitosan; acidic polymers obtainable from natural sources suchas alginic acid, hyaluronic acid, pectin, gum tragacanth, and karayagum; and neutral non-naturally occurring polymers, such aspolyvinylalcohol; or combinations thereof.

[0063] The phrase “immunostimulatory molecules”, as used herein, refersto those molecules that enhance the protective immune response inducedby an antigenic component in vaccine compositions. Suitableimmunostimulatory materials include bacterial cell wall components,e.g., derivatives of N-acetyl muramyl-L-alanyl-D-isoglutamine such asmurabutide, threonyl-MDP and muramyl tripeptide; saponin glycosides andderivatives thereof, e.g., Quil A, QS 21 and GPI-0100; cholesterol; andquaternary ammonium compounds, e.g., dimethyldioctadecylammonium bromide(DDA) and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine(“avridine”).

[0064] Saponis are glycosidic compounds that are produced as secondarymetabolites in a wide variety of plant species. The chemical structureof saponins imparts a wide range of pharmacological and biologicalactivities, including some potent and efficacious immunologicalactivity.

[0065] Structurally, saponins consist of any aglycone attached to one ormore sugar chains. Saponins can be classified according to theiraglycone composition: Triterpene glycosides, Steroid glycosides, andSteroid alkaloid glycosides.

[0066] Saponin can be isolated from the bark of Quillaja saponaria.Saponin has long been known as an immunostimulator. Dalsgaard, K.,“Evaluation of its adjuvant activity with a special reference to theapplication in the vaccination of cattle against foot-and-mouthdisease”, Acta. Vet. Scand. 69: 1-40 1978. Crude extracts of plantscontaining saponin enhanced potency of foot and mouth disease vaccines.However, the crude extracts were associated with adverse side effectswhen used in vaccines. Subsequently, Dalsgaard partially purified theadjuvant active component from saponin by dialysis, ion exchange and gelfiltration chromatography. Dalsgaard, K. et al., “Saponin adjuvants III.Isolation of a substance from Quillaja saponaria Morina with adjuvantactivity in foot-and-mouth disease vaccines”, Arch. Gesamte.Virusforsch. 44: 243-254 1974. An adjuvant active component purified inthis way is known as “Quil A.” On a weight basis Quil A showed increasedpotency and exhibited reduced local reactions when compared to crudesaponin. Quil A is widely used in veterinary vaccines.

[0067] Further analysis of Quil A by high pressure liquid chromatography(HPLC) revealed a heterogenous mixture of closely related saponins andled to discovery of QS21 which was a potent adjuvant with reduced orminimal toxicity. Kensil C. R. et al., “Separation and characterizationof saponins with adjuvant activity from Quillaja saponaria Molinacortex,” J. Immunol. 146: 431-437, 1991. Unlike most otherimmunostimulators, QS 21 is water-soluble and can be used in vaccineswith or without emulsion type formulations. QS21 has been shown toelicit a Th1 type response in mice stimulating the production of IgG2aand IgG2b antibodies and induced antigen-specific CD8+CTL (MHC class I)in response to subunit antigens. Clinical studies in humans have provedits adjuvanticity with an acceptable toxicological profile. Kensil, C.R. et al., “Structural and imunological charaterization of the vaccineadjuvant QS-21. In Vaccine Design: the subunit and Adjvuant Approach,”Eds. Powell, M. F. and Newman, M. J. Plenum Publishing Corporation, NewYork. 1995, pp. 525-541.

[0068] U.S. Pat. No. 6,080,725 teaches the methods of making and usingsaponin-lilpophile conjugate. In this saponin-lipophile conjugate, alipophile moiety such as lipid, fatty acid, polyethylene glycol orterpene is covalently attached to a non-acylated or desacylatedtriterpene saponin via a carboxy group present on the 3-O-glucuronicacid of the triterpene saponin. The attachment of a lipophilic moiety tothe 3-O-glucuronic acid of a saponin such as Quillaja desacylsaponin,lucyoside P, or saponin from Gypsophila, saponaria and Acanthophyllumenhances their adjuvant effects on humoral and cell-mediated immunity.Additionally, the attachment of a lipophile moiety to the 3-O-glucuronicacid residue of non- or desacylsaponin yields a saponin analog that iseasier to purify, less toxic, chemically more stable, and possessesequal or better adjuvant properties than the original saponin.

[0069] GPI-0100 is a saponin-lipophile conjugate described in the U.S.Pat. No. 6,080,725. GPI-0100 is produced by the addition of aliphaticamine to desacylsaponin via the carboxyl group of glucuronic acid.

[0070] Quaternary ammonium compounds—A number of aliphatic nitrogenousbases have been proposed for use as immunological adjuvants, includingamines, quaternary ammonium compounds, guanidines, benzamidines andthiouroniums. Specific such compounds includedimethyldioctadecylammonium bromide (DDA) andN,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine (“avridine”).

[0071] U.S. Pat. No. 5,951,988 teaches adjuvant formulation containingquarternary ammonium salts such as DDA in conjunction with an oilcomponent. This formulation is useful in conjunction with knownimmunological substances, e.g., viral or bacterial antigens in a vaccinecomposition, in order to enhance the immunogenic response. Thecomposition is also useful without an incorporated antigen asnonspecific immunostimulatory formulation.

[0072] U.S. Pat. No. 4,310,550 describes the use of N,N-higheralkyl-N,N′-bis(2-hydroxyethyl)-propanediamine and N,N-higheralkyl-xylylenediamines formulated with fat or lipid emulsion as avaccine adjuvant. A method of inducing or enhancing the immunogenicresponse of an antigen in man or an animal through parenteraladministration of the adjuvant formulation is described in the U.S. Pat.No. 4,310,550.

[0073] In a preferred embodiment, the present invention provides asubmicron oil-in-water emulsion useful as vaccine adjuvant, which iscomposed of an AMPHIGEN® formulation, with droplets of a size less than1 μm and a mean droplet size of about 0.25 μm.

[0074] The term “AMPHIGEN® formulation” as used herein refers to asolution formed by mixing a DRAKEOL® lecithin oil solution (Hydronics,Lincoln, NE) with saline solution in the presence of TWEEN® 80 and SPAN®80. A typical AMPHIGEN® formulation contains 40% light mineral oil byvolume (v/v), about 25% w/v lecithin, about 0.18% TWEEN 80 by volume(v/v) and about 0.08% Span 80 by volume (v/v).

[0075] Methods of Preparing Submicron Oil-In-Water Emulsions

[0076] In another embodiment, the present invention provides methods ofpreparing the submicron oil-in-water emulsions described hereinabove.

[0077] According to the present invention, the various components of theemulsion, including oil, one or more surfactants, an aqueous componentand any other component appropriate for use in the emulsion, arecombined and mixed together.

[0078] The mixture formed is subjected to an emulsification process,typically by passage one or more times through one or more homogenizersor emulsifiers to form an oil-in-water emulsion which has a uniformappearance and an average droplet size of about 0.5 μm. Any commerciallyavailable homogenizer or emulsifier can be used for this purpose, e.g.,Ross emulsifier (Hauppauge, N.Y.), Gaulin homogenizer (Everett, Mass.).

[0079] The emulsion so formed is then subjected to microfluidization tobring the droplet size in the submicron range. Microfluidization can beachieved by use of a commercial mirofluidizer, such as model number 11OY available from Microfluidics, Newton, Mass.; Gaulin Model 30CD(Gaulin, Inc., Everett, Mass.); and Rainnie Minilab Type 8.30H (MiroAtomizer Food and Dairy, Inc., Hudson, Wis.). These microfluidizersoperate by forcing fluids through small apertures under high pressure,such that two fluid streams interact at high velocities in aninteraction chamber to form emulsions with droplets of a submicron size.

[0080] Droplet size can be determined by a variety of methods known inthe art, e.g., laser diffraction, by use of commercially availablesizing instruments. The size may vary depending on the type ofsurfactant used, the ratio of surfactant to oil, operating pressure,temperature, and the like. The skilled artisan can determine the desiredcombination of these parameters to obtain emulsions with desired dropletsize without undue experimentation. The droplets of the emulsions of thepresent invention are less than 1 μm in diameter, preferably with a meandroplet size of less than 0.8 μm, and more preferably with a meandroplet size less than 0.5 μm, and even more preferably with a meandroplet size of less than 0.3 μm.

[0081] In a preferred embodiment of the present invention, the DRAKEOLlecithin oil solution, which is commercially available from Hydronics(Lincoln, Nebr.) and contains 25% lecithin in light mineral oil, iscombined and mixed with saline as well as surfactants TWEEN® 80 andSPAN® 80 to form an “AMPHGEN® solution” or “AMPHIGEN® formulation”. TheAMPHGEN® solution is then emulsified with a Ross® (Hauppauge, N.Y.11788) emulsifier at approximately 3400 rpm to form an oil-in-wateremulsion. Subsequently the emulsion is passed once through aMicrofluidizer operating at about 4500±500 psi. The microfluidizedoil-in-water emulsion has droplets of a size less than 1 μm, with a meandroplet size of about 0.25 μm.

[0082] Vaccine Compositions Containing Antigens Incorporated inSubmicron Oil-In-Water Emulsions

[0083] In another embodiment, the present invention provides vaccinecompositions which contain an antigen(s) and a submicron oil-in-wateremulsion described hereinabove. These vaccine compositions arecharacterized by having an enhanced immunogenic effect and an improvedphysical appearance (e.g., no phase separation is observed after anextended period of storage). In addition, the vaccine compositions ofthe present invention are safe for administration to animals.

[0084] According to the present invention, the antigen can be combinedwith the emulsion extrinsically, or preferably, intrinsically. The term“intrinsically” refers to the process wherein the antigen is combinedwith the emulsion components prior to the microfluidization step. Theterm “extrinsically” refers to the process where the antigen is added tothe emulsion after the emulsion has been microfluidized. Theextrinsically added antigen can be free antigen or it can beencapsulated in microparticles as further described herein below.

[0085] The term “antigen” as used herein refers to any molecule,compound or composition that is immunogenic in an animal and is includedin the vaccine composition to elicit a protective immune response in theanimal to which the vaccine composition is administered.

[0086] The term “immunogenic” as used in connection with an antigenrefers to the capacity of the antigen to provoke an immune response inan animal against the antigen. The immune response can be a cellularimmune response mediated primarily by cytotoxic T-cells, or a humoralimmune response mediated primarily by helper T-cells, which in turnactivates B-cells leading to antibody production.

[0087] A “protective immune response” is defined as any immune response,either antibody or cell mediated immune response, or both, occurring inthe animal that either prevents or detectably reduces the occurrence, oreliminates or detectably reduces the severity, or detectably slows therate of progression, of the disorder or disease caused by the antigen ora pathogen containing the antigen.

[0088] Antigens which can be included in the vaccine composition of thepresent invention include antigens prepared from pathogenic bacteriasuch as Mycoplasma hyopneumoniae, Haemophilus somnus, Haemophilusparasuis, Bordetella bronchiseptica, Actinobacillus pleuropneumonie,Pasteurella multocida, Manheimia hemolytica, Mycoplasma bovis,Mycoplasma galanacieum, Mycobacterium bovis, Mycobacteriumparatuberculosis, Clostridial spp., Streptococcus uberis, Streptococcussuis, Staphylococcus aureus, Erysipelothrix rhusopathiae, Campylobacterspp., Fusobacterium necrophorum, Escherichia coli, Salmonella entericaserovars, Leptospira spp.; pathogenic fungi such as Candida; protozoasuch as Cryptosporidium parvum, Neospora canium, Toxoplasma gondii,Eimeria spp.; helminths such as Ostertagia, Cooperia, Haemonchus,Fasciola, either in the form of an inactivated whole or partial cellpreparation, or in the form of antigenic molecules obtained byconventional protein purification, genetic engineering techniques orchemical synthesis. Additional antigens include pathogenic viruses suchas Bovine herpesviruses-1,3,6, Bovine viral diarrhea virus (BVDV) types1 and 2, Bovine parainfluenza virus, Bovine respiratory syncytial virus,bovine leukosis virus, rinderpest virus, foot and mouth disease virus,rabies, swine fever virus, African swine fever virus, Porcineparvovirus, PRRS virus, Porcine circovirus, influenza virus, swinevesicular disease virus, Techen fever virus, Pseudorabies virus, eitherin the form of an inactivated whole or partial virus preparation, or inthe form of antigenic molecules obtained by conventional proteinpurification, genetic engineering techniques or chemical synthesis.

[0089] The amount of the antigen should be such that the antigen which,in combination with the oil-in-water emulsion, is effective to induce aprotective immune response in an animal. The precise amount of anantigen to be effective depends on the nature, activity and purity ofthe antigen, and can be determined by one skilled in the art.

[0090] The amount of the oil-in-water emulsion present in the vaccinecompositions should be sufficient for potentiating the immunogenicity ofthe antigen(s) in the vaccine compositions. When desirable andappropriate, additional amounts of surfactant(s) or additionalsurfactant(s) can be added in the vaccine composition in addition to thesurfactant(s) provided by the oil-in-water emulsion. Generally speaking,the oil component is present in the final volume of a vaccinecomposition in an amount from 1.0% to 20% by volume; preferably, in anamount of 1.0% to 10%; more preferably, in an amount from 2.0% to 5.0%.The surfactant, or the combination of surfactants if two or moresurfactants are used, is present in the final volume of a vaccinecomposition in an amount of 0.1% to 20% by volume, preferably, 0.15% to10%, more preferably 0.2% to 6.0%.

[0091] In addition to the antigen(s) and the oil-in-water emulsion, thevaccine composition can include other components which are appropriateand desirable, such as preservatives, osmotic agents, bioadhesivemolecules, and immunostimulatory molecules (e.g., Quil A, cholesterol,GPI-0100, dimethyldioctadecylammonium bromide (DDA)), as describedhereinabove in connection with the oil-in-water emulsion.

[0092] The vaccine compositions of the present invention can alsoinclude a veterinarily-acceptable carrier. The term “aveterinarily-acceptable carrier” includes any and all solvents,dispersion media, coatings, adjuvants, stabilizing agents, diluents,preservatives, antibacterial and antifungal agents, isotonic agents,adsorption delaying agents, and the like. Diluents can include water,saline, dextrose, ethanol, glycerol, and the like. Isotonic agents caninclude sodium chloride, dextrose, mannitol, sorbitol, and lactose,among others. Stabilizers include albumin, among others.

[0093] In a preferred embodiment, the present invention provides avaccine composition which includes at least one of a BVDV type I or BVDVtype II antigen, incorporated intrinsically in an oil-in-water emulsionwhich has droplets of a size of less than 1 μm, preferably with a meandroplet size of less than 0.8 μm, more preferably less than 0.5 μm, andeven more preferably with a mean droplet size of about 0.5 μm. The BVDVtype I and/or II antigen is preferably in the form of an inactivatedviral preparation. The submicron oil-in-water emulsion preferably iscomposed of an AMPHIGEN® formulation (i.e., a formulation which containslight mineral oil, lecithin, TWEEN® 80, and SPAN® 80). The vaccinecomposition preferably also includes Quil-A, cholesterol, andthimerosol.

[0094] In another preferred embodiment, the present invention provides avaccine composition which includes a Leptospira antigen and at least oneof a BVDV type I or BVDV type II antigen in an oil-in-water emulsion.The antigens, preferably in the form of inactivated cell or viralpreparation, are incorporated intrinsically in the oil-in-water emulsionhaving droplets of a size of less than 1 μm, preferably with a meandroplet size of less than 0.8 μm, more preferably less than 0.5 μm, andeven more preferably with a mean droplet size of about 0.5 μm. Thesubmicron oil-in-water emulsion preferably is composed of an AMPHIGENformulation (i.e., a formulation which contains light mineral oil,lecithin, TWEEN® 80, and SPAN® 80). The vaccine composition preferablyalso includes one or more immunostimulatory molecules selected fromQuil-A, cholesterol, DDA, GPI-100 and aluminum hydroxide (AIOH).

[0095] In still another preferred embodiment, the present inventionprovides a vaccine composition which includes at least one bacterialantigen, e.g., the recombinant Streptococcus uberis PauA protein or acell preparation of E. coli or a combination of both, in an oil-in-wateremulsion. The antigen(s) is combined intrinsically with the oil-in-wateremulsion which has droplets of a size of less than 1 μm, preferably witha mean droplet size of less than 0.8 μm, more preferably less than 0.5μm, and even more preferably with a mean droplet size of about 0.25 μm.The submicron oil-in-water emulsion preferably is composed of anAMPHIGEN® formulation (i.e., a formulation which contains light mineraloil, lecithin, TWEEN® 80, and SPAN® 80). The vaccine compositionpreferably also includes one or more immunostimulatory moleculesselected from Quil A, DDA and GPI-100.

[0096] The vaccine compositions of the present invention can beadministered to an animal by known routes, including the oral,intranasal, mucosal, topical, transdermal, and parenteral (e.g.,intravenous, intraperitoneal, intradermal, subcutaneous orintramuscular) route. Administration can be achieved using a combinationof routes, e.g., first administration using a parental route andsubsequent administration using a mucosal route.

[0097] Methods of Preparing Vaccine Compositions

[0098] In a further embodiment, the present invention provides methodsof preparing vaccine compositions containing an antigen or antigens anda submicron oil-in-water emulsion.

[0099] In preparing the vaccine compositions of the present invention,the antigen(s) can be combined either intrinsically or extrinsicallywith the components of the oil-in-water emulsion. Preferably, theantigen is combined with the components of the oil-in-water emulsionintrinsically.

[0100] The antigen can be combined with the various components of theemulsion, including oil, one or more surfactants, an aqueous componentand any other appropriate component, to form a mixture. The mixture issubjected to a primary blending process, typically by passage one ormore times through one or more homogenizers or emulsifiers, to form anoil-in-water emulsion containing the antigen. Any commercially availablehomogenizer or emulsifier can be used for this purpose, e.g., Rossemulsifier (Hauppauge, N.Y.), Gaulin homogenizer (Everett, Mass.), orMicrofluidics (Newton, Mass.). Alternatively, the various components ofthe emulsion adjuvant, including oil, one or more surfactants, and anaqueous component can be combined first to form an oil-in-water emulsionby using a homogenizer or emulsifier; and the antigen is then added tothis emulsion. The mean droplet size of the oil-in-water emulsion afterthe primary blending is approximately 1.0-1.2 micron.

[0101] The emulsion containing the antigen is then subjected tomicrofluidization to bring the droplet size in the submicron range.Microfluidization can be achieved by use of a commercial mirofluidizer,such as model number 11OY available from Microfluidics, Newton, Mass.;Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.); and Rainnie MinilabType 8.30H (Miro Atomizer Food and Dairy, Inc., Hudson, Wis.).

[0102] Droplet size can be determined by a variety of methods known inthe art, e.g., laser diffraction, by use of commercially availablesizing instruments. The size may vary depending on the type ofsurfactant used, the ratio of surfactant to oil, operating pressure,temperature, and the like. One can determine a desired combination ofthese parameters to obtain emulsions with a desired droplet size. Theoil droplets of the emulsions of the present invention are less than 1μm in diameter. Preferably the mean droplet size is less than 0.8 μm.More preferably, the mean droplet size is less than 0.5 μm. Even morepreferably, the mean droplet size is about 0.1 to 0.3 μm.

[0103] In a preferred embodiment of the present invention, the DRAKEOL®lecithin oil solution, which contains 25% lecithin in light mineral oil,is combined and mixed with surfactants TWEEN® 80 and SPAN® 80 and salinesolution to form a mixture that contains 40% light mineral oil,lecithin, 0.18% TWEEN® 80, and 0.08% SPAN® 80. The mixture is thenemulsified with a Ross® (Hauppauge, N.Y. 11788) emulsifier atapproximately 3400 rpm to form an emulsion product, which is alsoreferred to as an “AMPHIGEN® formulation” or “AMPHIGEN® solution”.Subsequently, the desired antigen(s) are combined with the AMPHIGEN®solution and any other appropriate components (e.g., immunostimulatorymolecules) with the aid of an emulsifier, e.g., a Ross homogenizer, toform an oil-in-water emulsion containing the antigen(s). Such emulsionis passed once through a Microfluidizer operating at about 10000±500psi. The microfluidized oil-in-water emulsion has droplets of a size ofless than 1 μm, with the mean droplet size of about 0.25 μm.

[0104] In another preferred embodiment, prior to combining anoil-in-water emulsion (e.g., an AMPHIGEN® formulation) with a desiredantigen(s), the antigen(s) is combined with a saponin glycoside, e.g.,Quil A, to form a mixture. This antigen(s)-saponin mixture is subjectedto homogenization, e.g., in a homogenization vessel. A sterol, e.g.,cholesterol, is then added to the homogenized antigen(s)-saponinmixture. The mixture containing the antigen(s), saponin and sterol isthen subjected to further homogenization. The homogenizedantigen(s)-saponin-sterol mixture is then combined with an oil-in-wateremulsion (e.g., an AMPHIGEN) formulation) with the aid of a homogenizer,for example. The homogenized oil-in-water emulsion containing theantigen(s), saponin and sterol is then subjected to high pressurehomogenization, such as microfluidization.

[0105] Vaccine Compositions Containing Microencapsulated Antigens in aSubmicron Oil-in-Water Emulsion and Methods of Preparation

[0106] In still another embodiment, the present invention providesvaccine compositions which contain an antigen encapsulated inmicroparticles (or “microencapsulated antigen”), where themicroencapsulated antigen is extrinsically incorporated into a submicronoil-in-water emulsion described hereinabove.

[0107] Methods for absorbing or entrapping antigens in particulatecarriers are known in the art. See, e.g., Pharmaceutical ParticulateCarriers: Therapeutic Applications (Justin Hanes, Masatoshi Chiba andRobert Langer. Polymer microspheres for vaccine delivery. In: Vaccinedesign. The subunit and adjuvant approach. Eds. Michael F. Powell andMark J. Newman, 1995 Plenum Press, New York and London ). Particulatecarriers can present multiple copies of a selected antigen to the immunesystem in an animal subject and promote trapping and retention ofantigens in local lymph nodes. The particles can be phagocytosed bymacrophages and can enhance antigen presentation through cytokinerelease. Particulate carriers have also been described in the art andinclude, e.g., those derived from polymethyl methacrylate polymers, aswell as those derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. Polymethyl methacrylatepolymers are non-biodegradable while PLG particles can be biodegrade byrandom non-enzymatic hydrolysis of ester bonds to lactic and glycolicacids which are excreted along normal metabolic pathways.

[0108] Biodegradable microspheres have also used to achieve controlledrelease of vaccines. For example, a continuous release of antigen over aprolonged period can be achieved. Depending upon the molecular weight ofthe polymer and the ratio of lactic to glycolic acid in the polymer, aPLGA polymer can have a hydrolysis rate from a few days or weeks toseveral months or a year. A slow, controlled release may result in theformation of high levels of antibodies similar to those observed aftermultiple injections. Alternatively, a pulsatile release of vaccineantigens an be achieved by selecting polymers with different rates ofhydrolysis. The rate of hydrolysis of a polymer typically depends uponthe molecular weight of the polymer and the ratio of lactic to glycolicacid in the polymer. Microparticles made from two or more differentpolymers with varying rates of antigen release provide pulsatilereleases of antigens and mimics multiple-dose regimes of vaccination.

[0109] According to the present invention, an antigen, including any ofthose described hereinabove, can be absorbed to a particulate polymercarrier, preferably a PLG polymer, by using any procedure known in theart (such as one exemplified in Example 17), to form a microencapsulatedantigen preparation. The microencapsulated antigen preparation is thenmixed with and dispersed in a submicron oil-in-water emulsion, whichemulsion has been described hereinabove, to form the vaccinecomposition.

[0110] In a preferred embodiment, the present invention provides avaccine composition which contains an antigen encapsulated in a PLGpolymer, wherein the microencapsulated antigen is dispersedextrinsically in a microfluidized oil-in-water emulsion which iscomposed of light mineral oil, lecithin, TWEEN80, SPAN80 and saline, andhas a mean droplet size of less than 1.0 μm.

[0111] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

EXAMPLE 1 Preparation of an AMPHIGEN® Formulation

[0112] An AMPHIGEN® formulation was prepared in a two-step process. Inthe first step, 80 liters of Drakeol Lecithin oil solution, 116 litersof Tetanus Toxoid saline, 1.2 liters of SPAN 80, and 2.8 liters of Tween80 were mixed together and emulsified using a Ross emulsifier. TheDrakeol Lecithin oil solution contained 25% soya lecithin and 75%mineral oil. Emulsified product was recirculated through Ross emulsifierfor a minimum of 5 volumes or a minimum of 10 minutes. The emulsifiedproduct was stored at 2-7° C. for a maximum of 24 hours for furtherprocessing. The emulsion from the Ross emulsifier tank was transferredto a Gaulin homogenizer and was homogenized for 20 minutes under apressure of 4500 psi. The resulting 40% Drakeol Lecithin oil solution(hereinafter the “AMPHIGEN®) formulation” or “AMPHIGEN® solution”) wasthen dispensed into sterile polypropylene carboxy containers. Thedispensing was performed inside a class 100 dispensing hood located in aclass 10,000 controlled environment. The containers were stored at 2-7°C. This AMPHIGEN® formulation was used in the experiments describedhereinbelow unless indicated otherwise.

EXAMPLE 2 Primary Blending by Flashblend Homogenization of the BVDVaccine

[0113] The apparatus used for this homogenization process is shown inFIG. 1. Using aseptic technique or steam cross valves, a bottlecontaining an BVD Type I antigen (an inactivated BVD Type I viralpreparation) was attached to the bottom side port on the blend vessel.After the transfer of required volume of the BVD Type I antigen wascompleted, the BVD Type I bottle was replaced with the bottle containingan inactivated BVD Type II viral preparation (an inactivated BVD type IIviral preparation). After the required amount of a BVD Type II antigentransfer was completed, the Ross homogenizer was attached to theportable vessel and the recirculation was initiated at maximum RPM (3300rpm). Vessel agitation was maintained at medium speed.

[0114] Using aseptic technique or stream cross valve, a bottlecontaining Quil-A at 50 mg/ml concentration was attached to thehomogenizer in-line port on the blend vessel. A required amount of theQuil-A solution was passed into the vessel through line suction. Afterthe transfer of the Quil-A solution was completed, the bottle wasremoved. In the same way, a required amount of cholesterol in ethanolsolution (18 mg/ml) was transferred to the blend vessel. Subsequently, arequired amount of the AMPHIGEN® formulation, 10% thimerosol solution,and Basic Modified Eagles media (“BME”) extender solutions were added tothe blend vessel.

[0115] Once all the additions were complete, the mixing was continuedfor an additional 15 minutes. The resulting formulation was aliquotedinto 2 ml doses and represented a non-microfluidized AMPHIGEN®formulation-based BVD vaccine. Each dose of the vaccine contained 500 μgQuil-A, 500 μg Cholesterol, 2.5% AMPHIGEN® formulation and 0.009%thimerosol. The antigen concentration for the two different BVD strainswas determined in terms of the ELISA titer for gp53.

EXAMPLE 3 Secondary Blending by Microfluidization

[0116]FIG. 2 illustrates the process used for the secondary blendingthrough microfluidization. The microfluidizer was steam sterilized.First the auxiliary processing module chamber was installed in the unitand the blank chamber was installed on the second chamber position. Thevessel containing the fully adjuvanted BVD vaccine prepared as describedin the Example 2 was connected to the microfluidizer by attaching atransfer line from the supply vessel drain valve to the microfluidizerinlet. Nitrogen gas was connected to the supply vessel air filter inletand the vessel pressure setting was adjusted to 20+/−5 PSI. Collectionvessel drain valve was connected to the transfer line from themicrofluidizer outlet. After making all the necessary connections, thevalves were opened and microfluidization was initiated at an operatingpressure of 10,000+/−500 PSI. The entire content of the vaccine waspassed through the microfluidizer one time and was collected in thepost-microfluidization chamber. This preparation was aliquoted into 2 mLdoses and represents the microfluidized AMPHIGEN® formulation-based BVDvaccine.

EXAMPLE 4

[0117] Preparation of a Vaccine Composition through Bench Blend.

[0118] The AMPHIGEN® formulation prepared as described in Example 1 wasdiluted to the 2.5% with the addition of BVD antigens and the extender.The resulting solution was blended at the bench using a stir bar insteadof using a homogenizer. The final preparation contained the followingcomposition: BVD Type 1 and Type 2 antigens, 2.5% AMPHIGEN® formulation(which contains oil, lecithin, SPAN® and TWEEN®, as described in Example1), and saline. TWEEN 80 and SPAN 80 are present in the final vaccinepreparation at 0.18% and 0.08% by volume, respectively.

EXAMPLE 5 Comparison of Droplet Size Distribution between theNon-Microfluidized and Microfluidized AMPHIGEN® Formulation-BasedVaccine Preparations

[0119] The non-microfluidized AMPHIGEN® formulation-based vaccineprepared as described in the Example 2, the microfluidized AMPHIGEN®formulation-based vaccine prepared as described in the Example 3, andthe preparation made through bench blend as described in Example 4, wereused to compare the droplet size of the vaccine preparations. Twomililiters of the sample from each of the preparations were added to aMalvern 2000 Laser Diffraction meter and the droplet size distributionwas determined. As shown in FIG. 3, the results indicate that themicrofluidized AMPHIGEN® formulation-based vaccine preparation had themaximum particle volume around 0.1 micron while the non-microfluidizedAMPHIGEN® formulation-based vaccine preparation had the maximum particledistribution volume around 1 micron.

EXAMPLE 6 Reduction in Vaccine Phase Separation

[0120] Three different vaccine preparations: the non-microfluidizedAMPHIGEN® formulation-based vaccine prepared as described in the Example2, the microfluidized AMPHIGEN® formulation-based vaccine prepared asdescribed in the Example 3, and the vaccine prepared through bench blendas described in Example 4, were compared side by side to determine theirphase separation properties upon long storage. All these preparationswere allowed to stand at 4° C. for about one month and the phaseseparation was monitored in terms of appearance of a creamy layer at thetop of the vaccine preparations. As shown in FIG. 4, there was no phaseseparation in the microfluidized AMPHIGEN® formulation-based preparationwhen compared to the other two preparations.

EXAMPLE 7 Preparation of Microfluidized and Non-Microfluidized CattleVaccine against Bovine Viral Diarrhea Virus

[0121] Bovine Virus Diarrhea viral antigen was intrinsicallyincorporated into the AMPHIGEN® formulation through microfluidization.The term “intrinsically incorporated” refers to the process whereby theantigen was added to the AMPHIGEN® formulation prior to themicrofluidization. The antigen was subjected to the physical forces ofthe microfluidization process along with the components of the adjuvantformulation. In the control non-microfluidized group, the antigenpreparation was dispersed in the AMPHIGEN® formulation through blending.

[0122] The final composition of both the control and microfluidizedpreparations was as follow: BVD type I with a post-inactivation ELISAtiter of 2535 RU/ dose for gp53, BVD Type II with a post-inactivationELISA titer of 3290 RU/dose for gp53, Quil-A at the concentration of1.25 mg/dose, cholesterol at the concentration of 1.25 mg/dose, theAMPHIGEN® formulation at the final concentration of 2.5%, and thimerosolat the final concentration of 0.009%. The vaccine dose was 5 ml.

EXAMPLE 8 Long Term Stability of Intrinsically Incorporated BVD ViralAntigens in the Microfluidized AMPHIGEN® Formulation-Based VaccinePreparation

[0123] This experiment was carried out to determine the stability of theintrinsically incorporated antigen during the long storage. Killed BVDType II viral antigen was intrinsically incorporated into the AMPHIGEN®formulation during microfluidization process to obtain microfluidizedvaccine preparation (A907505). Three other vaccine preparationscontaining the same antigen in non-microfluidized AMPHIGEN® formulation(A904369, A904370, and A904371) served as the control. In thenon-microfluidized preparations, the antigen was mixed with AMPHIGEN®formulation and mixed through blending using a Ross homogenizer. Allfour vaccine preparations were stored at 4° C. for two years. Atdifferent points during the storage (0, 6, 12 or 24 months), all fourformulations were used to vaccinate three months old cows.

[0124] On days 0 and 21, three-month old cows were vaccinated throughsubcutaneous route with a 2 ml vaccine formulation. The serum from thevaccinated animals was collected on day 35, and serological response tothe vaccine was measured in terms of the antibody titer through BVDV-E2ELISA. As shown in FIG. 5, the microfluidized vaccine preparation showeda higher antibody titer at all the time points tested (0, 6, 12, and 24months), suggesting the stability of the antigen preparation is not lostduring the intrinsic incorporation of the antigen during themicrofuidization process. Moreover, it was also surprisingly found thatthe microfluidized vaccine preparation induced an enhanced immuneresponse at all time points.

EXAMPLE 9 Reduction in the Vaccine-Induced Increase in RectalTemperature after Microfluidization

[0125] The microfluidized and non-microfluidized vaccine preparationsmade as described in Example 7 were used to vaccinate the cattle on dayzero and the rectal temperature was monitored during the period from oneday prior to vaccination till four days post vaccination. The vaccinedose was 2 ml. The groups were vaccinated either with a single or doubledose of the vaccine. Rectal temperatures were measured and recordeddaily on Day -1 through Day 4, inclusive. Rectal temperatures on day 0were measured prior to administration of test article.

[0126] As shown in FIG. 6, the results indicate that there was a steeprise in the rectal temperature in about 24 hours following vaccinationin those animals vaccinated with either a single or double dose of thenon-microfluidized vaccine formulation. However, in the animalsvaccinated with microfluidized forms of vaccine, the rise in rectaltemperature following the vaccination was only minimal and significantlylower than in the animals vaccinated with the non-microfluidizedformulation (FIG. 6).

EXAMPLE 10 The Injection Site Reaction Volume was Resolved Faster whenVaccinated with Microfluidized Vaccine Formulations

[0127] The microfluidized and non-microfluidized vaccine preparationsmade as described in the Example 7 were used to vaccinate the cattle onday zero. The animals included in this study were cross-bred beefcattle. There were three animals in each of the placebo treatment groups(T01 and T02). There were six animals in each of the groups T03 throughT06. The vaccine dose was 2 ml and the groups were vaccinated eitherwith one or two doses of the vaccine on day zero. On day 0, test articlewas administered in the right neck. Animals receiving the double dose (4ml) of the test article (T02, T04, and T06) received the entire doubledose as a single injection at one side. Observation of injection sites,including estimation of reaction size at the injection site were made onthe right side of the neck on Day 0 through Day 4, inclusive, and Days6, 9, and 14. On Day 0 injection sites were observed prior toadministration of test articles. The groups vaccinated with one or twodoses of the placebo did not show any significant increase in theinjection site reaction volume and therefore those data are not shown inthe FIG. 7. In the case of the non-microfluidized vaccine formulation,there was a proportional increase in the injection site reaction volumebetween the one dose and two dose vaccination. On the other hand, in thecase of the microfluidized vaccine formulation, although the single doseinduced a larger injection site reaction volume, the injection withsecond dose did not cause any further increase. Moreover, in the case ofthe animals injected with microfluidized vaccine formulation, theinjection site reaction site volume was resolved at a faster rate whencompared to that in the animals injected with a non-microfluidizedvaccine formulation. These results are shown in FIG. 7.

EXAMPLE 11 Preparation of Microfluidized AMPHIGEN® Formulation-BasedVaccine Preparations with Intrinsically Incorporated BVD Viral andLeptospira Antigens and Immunostimulatory Molecules Such as Quil A andDDA

[0128] Formalin-inactivated Leptospira hardjo-bovis strain CSL wasformulated in the appropriate adjuvant at direct counts of about 1.4×10⁹organisms/5 ml dose. Formalin-inactivated Leptospira Pomona strain T262was formulated at about 2400 Nephalomeric Units/5 ml dose. Nephalomericunits were calculated based on nephalometric measurement of preprocessedfermentation fluid. BVD virus Type 1 was formulated at E2 Elisa titer ofabout 3000 Relative Units/5 ml dose. BVD virus Type 2 was formulated atE2 Elisa titer of about 3500 Relative Units/5 ml dose. The Relative Unitwas calculated based on the E2 ELISA titer of pre-assemblypost-inactivation bulk fluid. Both Quil-A and cholesterol were used atthe concentration of 0.5 mg per dose. Thimerosol and the AMPHIGEN®formulation were used at the final concentration of 0.009% and 2.5%,respectively. Aluminum hydroxide (Rehydragel LV) was used at the finalconcentration of 2.0%. When DDA was used as an immunomodulator, DDA wasincluded within the AMPHIGEN® formulation. The AMPHIGEN® formulation(i.e., the 40% Drakeol-lecithin stock solution) contained 1.6 mg/ml ofDDA and, when appropriately diluted, the final vaccine preparationcontained 2.5% AMPHIGEN® formulation and 0.1 mg/ml of DDA.

[0129] In the preparation of different vaccine formulations, BVDfractions, Leptos, Quil-A, chloestrol, thimerosol, the AMPHIGEN®formulation, and saline as an extender were added to a Silversonhomogenizer and mixed for 15 minutes at 10,000±500 RPM. Components werethen microfluidized through a 200 micron screen at 10,000 psi.

[0130] When the vaccine formulation contained aluminum hydroxide, themicrofluidization was carried out without aluminum hydroxide. After themicrofluidization was completed, aluminum hydroxide was added and mixedwith a stir bar overnight at 4° C.

EXAMPLE 12 Preparation of BVD Viral Vaccine for Challenge Studies

[0131] The vaccine preparation used in this experiment containedantigens from both BVD virus Type 1 and BVD Virus Type 2. BVD1 -5960antigen was used at the post-inactivation ELISA titer of 2535 RU/dosefor gp53. BVD2-890 antigen was used at the post-inactivation ELISA titerof 3290 RU/dose for gp53. Quil A and cholesterol were used at theconcentration of 0.5 mg/ml. Thimersol and the AMPHIGEN® formulation wereused at the final concentration of 0.009% and 2.5%, respectively. WhenDDA was used as an immune modulator, DDA was included within the theAMPHIGEN® formulation. The AMPHIGEN® stock solution (40%Drakeol-lecithin solution) contained varying amounts of DDA and whenappropriately diluted, the final vaccine preparation contained 2.5%AMPHIGEN®) formulation and DDA concentration ranging from 0.5 mg/dose to2.0 mg/dose. Aluminum gel (Rehydragel-LV) was used at the finalconcentration of 2%. GPI-0100 was used in the range of 2, 3, and 5mg/dose.

[0132] All the components were added to a Silverson homogenizer andblended for 15 minutes at 10,500 rpm and then microfluidized by passingthrough a 200 micron chamber with 10,000 psi. When the vaccinepreparation contained aluminum hydroxide, the microfluidization wascarried out without aluminum hydroxide. After the microfluidization wascompleted, aluminum hydroxide was added and mixed with a stir barovernight at 4° C.

EXAMPLE 13 Protection against Leptospira Challenge after Vaccinationwith a Microfluidized Amphigen Vaccine Formulation with LeptospiraAntigens

[0133] TABLE 1 Treatment Groups Treatment group Composition of adjuvantT01 Salilne T02 Quil-A, Cholesterol, and the AMPHIGEN ® formulation(QAC) T03 Quil-A, Cholesterol, the AMPHIGEN ® formulation and AIOH(QAC-AIOH) T04 DDA, Cholesterol, and the AMPHIGEN ® formulation (DDA)T05 DDA, Cholesterol, the AMPHIGEN ® formulation, and AIOH (DDA-AIOH)

[0134] Table 1 shows the composition of the adjuvant formulations in thevaccine preparations tested in this study. The vaccine preparations wereprepared as described in the Example 11. There were six animals in eachgroup. About seven-month old beef cross-bred heifers were used in thisstudy. Vaccination was done on Day 0 and Day 21 through subcutaneousroute with 5 ml vaccine volume. Challenge was done with L. hardjo-bovisstrain 203 from NADC (National agricultural Disease Center). Challengewas done during Days 57-59 with a 1-ml innoculum. Challenge wasadministered conjunctively in the eye and vaginally. The challengematerial contained 5.0×10⁶ leptospires/ml. Urine was collected weeklyfor lepto culture, FA and PCR. Kidney collection was made during Days112 and 113. TABLE 2 Results of the Leptospira Challenge Study PercentPercent of calves ever Percent of Percent of Calves ever positive forcalves ever calves ever positive for Leptospira positive for positivefor Leptospira in urine Leptospira Leptospira in Urine and Kidney inurine in urine and Kidneys through and Kidney and Kidney acrossTreatment Culture through FA through PCR all assays Saline 100 83.3 83.3100 QAC 0 0 0 0 QAC/AIOH 0 50.0 0 50.0 DDA 0 0 0 0 DDA/AIOH 0 33.3 16.750.0

[0135] Table 2 shows the data from the Leptospira challenge study. Indetermining the percentage of Leptospira infection in the challengedanimal, the following criteria were used. If the kidney culture waspositive for only one sample, the animal is considered to be positivefor Leptospira. If an animal is positive in only one sample for eitherFA or PCR, the animal is considered to be negative. If the sample ispositive for both FA and PCR in only one sample, it was consideredpositive for Leptospira.

[0136] The results shown in Table 2 indicate that there was asignificant shorter duration of urinary shedding in all vaccine groupsbased on all the three assays. As far as urinary and kidney colonizationare concerned, the efficacies of the QAC- and DDA-containingformulations without AIOH were comparable. AIOH did not improve and evenreduced the efficacies of the QAC- or DDA-containing vaccines in thischallenge study. TABLE 3 Microscopic Agglutination Titer Range On Day OfPeak Geometric Mean Titer Prior To Challenge (Day 35) Treatment L.Hardjo L. pomona Saline <20 <20 QAC 160-640 1280-10240 QAC/AIOH 160-2560   8-10240 DDA  40-1280  320-2560 DDA/AIOH 320-640 1280-5120

[0137] Serological responses against both of the Leptospira antigens inthe vaccine formulation ere detected in the vaccinated animal and thepeak response was noted on Day 35. There was no correlation between theserological response and the protection against the challenge. Thepresence of aluminum gel in the vaccine formulation reduced the level ofprotection although the serological response was enhanced by thepresence of aluminum gel in the vaccine.

EXAMPLE 14 Elicitation of Immune Response to the BVD Viral Antigen andProtection against the BVD Type 2 Virus Challenge after Immunizationwith a Microfluidized Vaccine Preparation Containing AMPHIGEN®Formulation and DDA

[0138] Four to seven month-old seronegative calves were used in thisexperiment. There were six different groups and each group had tenanimals (Table 4). On Day 0 and Day 21 each animal received one 2 mlsubcutaneous dose of the vaccine or placebo in the lateral neckapproximately midway between the scapula and poll. TABLE 4 TreatmentGroups Treatment Adjuvant composition T01 Saline T02 Quil-A, AMPHIGEN ®formulation, and Chloesterol T03 AMPHIGEN ® formulation, Choloesterol,DDA (0.5 mg/dose) and AIOH T04 AMPHIGEN ® formulation, Cholesterol, andDDA (0.5 mg/dose) T05 AMPHIGEN ® formulation, Cholesterol, and DDA (1.0mg/dose) T06 AMPHIGEN ® formulation, Cholesterol, and DDA (2.0 mg/dose)

[0139] A 5 ml dose of the challenge virus preparation (approximately 2.5ml per nostril) was administered intranasally on Day 44 of the study.Noncytopathic BVD virus Type 2, isolate # 24515 (Ellis Strain), lot #46325-70 was used in this study as the challenge strain. Retainedsamples of challenge material were tittered (two replicates pertitration) at the time challenge was initiated and immediately upon itscompletion. The mean live virus titer per 5 ml dose was 5.3 log₁₀FAID₅₀/5 ml prior to challenge and 5.4 log₅₀ FAID₅₀/5 ml post challenge(FAID is equivalent to TCID₅₀).

[0140] Animals were monitored daily from Day -3 through Day 58. Clinicaldisease scores of 0, 1, 2, or 3, based on clinical signs attributable toBVD 2 infection were made for each animal on Days 42 through 58. Thescores on Day 44 were recorded prior to challenge. Blood samples (two 13ml Serum Separation Tubes, SST) were collected from each animal on Days0, 21, 35, 44, and 58 for determination of serum titers of BVD Type 1and BVD Type 2 virus neutralization antibodies.

[0141] Blood samples were collected from each animal on Days 42 throughDay 58, inclusive, and the presence of BVD virus in buffy coat cell wasdetermined. On Day 44, samples were obtained prior to challenge.

[0142] For determining white blood cell counts, blood samples (one 4 mlEDTA tube) were collected from each animal on Day 42 through Day 58,inclusive. On Day 44, samples were obtained prior to challenge.

[0143] Leukopenia was defined as a 40% or greater decrease in the WBCcount from baseline (average of pre-challenge WBC counts from two daysprior to, and the day of challenge).

[0144] Clinical disease scores were used to define disease status asfollows; if the score is ≦1, then disease=no; if the score is >2, thendisease=yes.

[0145] As shown in the Tables 5 and 6, the groups vaccinated withvaccines containing BVD viral antigens along with the AMPHIGEN®)formulation, Quil A or DDA and microfluidized, seroconverted withsignificant serum virus neutralization titers for both BVD Type 1 andBVD Type 2 viruses. In those groups there was also a significantreduction in the percentage of animals showing viremia followingchallenge, while in the control group 100% of the animals were viremic(Table 7). In addition, in those vaccinated groups the frequency of thedisease was also significantly reduced (Table 8). Similarly, thepercentage of animals showing leukopenia was also reduced in the vaccinegroups and the reduction of leukopenia was more significant in the groupcontaining DDA than in the group containing Quil A (Table 9). In thecontrol group there was a significant drop in the weight gain whencompared to the vaccinated groups. (Table 10)

[0146] Serology

[0147] Prior to vaccination on Day 0, all animals in the study wereseronegative (SVN<1:2) for antibodies to BVD virus Types 1 and 2 (datanot shown). Fourteen days after the second vaccination (Day 35), allanimals that were administered the placebo (T01) remained seronegativefor antibodies to BVD virus Types 1 and 2; and all of the animalsvaccinated with the ITAs (Investigational Test Antigen) (T02, T03, T04,T05 and T06) were seropositive (SVN≧1:8) for antibodies to BVD virus,Types 1 and 2. One animal which was administered with the vaccineadjuvanted with the AMPHIGEN® formulation at 2 mg/dose of DDA had an SVNtiter of 3 for antibodies to BVD virus Type 2 on Day 35 (Table 11 and12).

[0148] Prior to challenge on Day 44, all controls (T01), except one,were seronegative (SVN<1:2) for antibodies to BVD virus Types 1 and 2(data now shown). The one control (#2497) was seropositive (SVN=10) forantibodies to BVD virus Type 1 and seronegative for antibodies to BVDvirus Type 2. Fourteen days following challenge, all animals in thestudy were seropositive for antibodies to BVD virus Types 1 and 2. TABLE5 BVD Virus Type 1 Geometric Mean Serum Virus Neutralization Titers BVDvType 1 Geometric Mean SVN Titers on Study Day Treatment 0 21 35 44 58T01 Saline <2 <2 <2 <2 23.9 T02 Amphigen, <2 39.1 19824.5 14018.227554.5 Quil A T03 Amphigen, <2 51.8 32204.8 22381.1 23170.4 0.5 mg DDA,Al T04 Amphigen, <2 27.0 14512.4 8932.0 21996.2 0.5 mg DDA T05 Amphigen,<2 26.7 11585.2 8194.6 20882.0 1.0 mg DDA T06 Amphigen, <2 23.5 8778.76769.3 16961.1 2.0 mg DDA

[0149] TABLE 6 BVD Virus Type 2 Geometric Mean Serum VirusNeutralization Titers BVDv Type 1 Geometric Mean SVN Titers on Study DayTreatment 0 21 35 44 58 T01 Saline <2 <2 <2 <2 522.0 T02 Amphigen, <28.9 2272.4 2048.2 24833.6 Quil A T03 Amphigen, <2 9.5 3565.7 2702.220881.8 0.5 mg DDA, Al T04 Amphigen, 0.5 mg <2 4.1 1260.7 989.1 18496.2DDA T05 Amphigen, <2 6.4 1398.8 1453.9 30047.8 1.0 mg DDA T06 Amphigen,2.0 mg <2 7.7 1673.2 1428.9 16384.0 DDA

[0150] TABLE 7 BVD Virus Isolation Following Challenge BVD VirusIsolation Frequency LSMean (%) of Viremic Days with Treatment On StudyDays Animals Viremia T01 Saline 47 through 58 10/10 (100.0) 10.4 T02Amphigen, 50 through 53 1/10 (10.0) 0.4 Quil A T03 Amphigen, 0.5 mg —0/10 (0.0)  0.0 DDA, Al T04 Amphigen, 48, 50 through 3/10 (30.0) 0.5 0.5mg DDA 52, 57 T05 Amphigen, 1.0 mg 49 through 51 2/10 (20.0) 0.4 DDA T06Amphigen, 2.0 mg 48 through 52 2/10 (20.0) 0.5 DDA

[0151] TABLE 8 Clinical Signs Of BVD Disease Following ChallengeFrequency Frequency (%) Observations with (%) with Clinical Sign of BVDDisease Total Treatment Disease 0 1 2 3 Obs. T01 Saline 9/10 (90.0) 75(46)  63 (37.5)   29 (17.3)   1 (0.6) 168 T02 Amphigen, 1/10 (10.0) 105(61.8) 63 (37.1)   2 (1.2) 0 (0) 170 Quil A T03 Amphigen, 2/10 (20.0) 99 (58.2) 67 (39.4)   4 (2.4) 0 (0) 170 0.5 mg DDA, Al T04 Amphigen,0/10 (0.0)  118 (69.4) 52 (30.6) 0 (0) 0 (0) 170 0.5 mg DDA T05Amphigen, 0/10 (0.0)  101 (59.4) 69 (40.6) 0 (0) 0 (0) 170 1.0 mg DDAT06 Amphigen, 0/10 (0.0)  104 (61.2) 66 (38.8) 0 (0) 0 (0) 170 2.0 mgDDA

[0152] TABLE 9 Leukopenia Following Challenge Leukopenia Frequency (%)of LSMean Days with Treatment Leukemic Animals Leukemia T01 Saline 10/10(100.0) 7.8 T02 Amphigen, Quil A 6/10 (60.0) 1.2 T03 Amphigen, 0.5 mg2/10 (20.0) 0.2 DDA, Al T04 Amphigen, 0.5 mg 4/10 (40.0) 0.8 DDA T05Amphigen, 3/10 (30.0) 0.9 1.0 mg DDA T06 Amphigen, 2.0 mg 2/10 (30.0)0.5 DDA

[0153] TABLE 10 Body Weight and Body Weight Gain During the Study MeanBody Weight (lb.) on Study Day Weight Treatment −1 43 50 58 Gain (lb)T01 Saline 378.0 484.9 491.0 476.9 98.9 T02 Amphigen, 428.0 526.5 546.7579.0 151.0 Quil A T03 Amphigen, 410.5 514.4 534.2 579.0 168.5 0.5 mgDDA, AlOH T04 Amphigen, 373.7 472.3 492.6 538.1 164.4 0.5 mg DDA T05Amphigen, 358.9 451.4 478.9 507.1 148.2 1.0 mg DDA T06 Amphigen, 408.513.3 533.9 560.3 151.6 2.0 mg DDA

[0154] Virus Isolation

[0155] As the data shown in Table 13, during the challenge period (Days44 through 58), all ten animals in the control (T01) were viremic (BVDvirus was isolated on one or more days). In the groups administered withthe ITAs, the frequency of viremic animals was one, zero, three, two andtwo in each group of ten (T02, T03, T04, T05 and T06, respectively). Thedifference between the control and the groups administered with the ITAswas statistically significant (P≦0.05). The least squares mean number ofdays of viremia was also significantly greater (10.4 days) for thecontrol as compared to the groups administered with the ITAs (0.0 to 0.5days).

[0156] Clinical Disease

[0157] Animals with clinical sign scores of 2 or 3 were considereddemonstrating signs of BVD disease. As shown in the Table 14, thefrequency of animals with clinical signs of BVD virus disease was nineof ten in the control (T01) and one, two, zero, zero and zero of ten ineach of the groups administered the ITAs (T02, T03, T04, T05 and T06,respectively). The difference between the control and groups that wereadministered the ITAs was statistically significant (P≦0.05).

[0158] Leukopenia

[0159] As shown in Table 15, during the challenge period (Days 44through 58), all ten animals in the control (T01) were leukemic (a 40%reduction in white blood cell count from pre-challenge baseline, Days42-44). The frequency of animals with leukemia was six, two, four, threeand two of the ten animals in each of the groups administered with theITAs (T02, T03, T04, T05 and T06, respectively). The difference betweenthe control and the group administered with vaccine which was adjuvantedwith the AMPHIGNEN®) formulation at 0.5 mg/dose and aluminum hydroxide(T03) was statistically significant (P≦0.05). The least squares meannumber of days of leukemia was significantly greater (7.8 days) for thecontrol as compared to the groups administered with the ITAs (0.2 to 1.2days).

EXAMPLE 15 Elicitation of Immune Response to the BVD Viral Antigen andProtection against the BVD Type 2 Virus Challenge after Immunizationwith Microfluidized Vaccine Formulation Containing GPI-0100

[0160] A set of experimental conditions as described in the Example 14was followed and a direct comparison between Quil A and GPI-0100 wasmade. As shown in the Tables 11 and 12, the animals vaccinated with BVDantigens in the microfluidized AMPHIGEN®) formulation-based preparationcontaining either Quil A or GPI-0100 had a significant antibody titerboth for BVD Type 1 and BVD Type 2 viruses. The antibody titer for BVDType 1 virus was much more higher than that for BVD Type 2 virus.However, subsequent challenge with BVD Type 2 virus showed a strongprotection and the disease incidence was significantly reduced in thecalves vaccinated with the microfluidized AMPHIGEN® formulation-basedvaccine preparation containing GPI-0100. TABLE 11 BVD virus Type 1Geometric Mean Serum Virus Neutralization Titers Geometric mean SVNtiter Treatment 0 21 35 43 57 T01 Saline <2 <2 <2 <2 35.5 T02 Amphigen,Quil A <2 98.7 20171.0 12203.4 44762.4 T03 Amphigen, 2 mg <2 84.610998.5 7383.2 25709.2 GPI-0100, AlOH T04 Amphigen, 2 mg <2 106.018179.2 8933.2 28526.2 GPI-0100 T05 Amphigen, 3 mg <2 62.9 15024.38780.1 19824.4 GPI-0100 T06 Am,phigen, 5 mg <2 71.1 12203.3 7512.016670.2 GPI-0100

[0161] TABLE 12 BVD virus Type 2 Geometric Mean Serum VirusNeutralization Titers BVDv Type 1 Geometric Mean SVN Titers on Study DayTreatment 0 21 35 44 58 T01 Saline <2 <2 <2 <2 14.7 T02 Amphigen, Quil A<2 12.9 2312.0 1692.5 1663.4 T03 Amphigen, 2 mg <2 13.2 1663.5 1116.81562.3 GPI-0100, AlOH T04 Amphigen, 2 mg <2 20.5 2610.2 1978.2 2478.7GPI-0100 T05 Amphigen, 3 mg <2 11.4 1752.8 1305.2 2435.4 GPI-0100 T06Amphigen, 5 mg <2 12.0 3158.4 2120.2 1845.6 GPI-0100

[0162] TABLE 13 BVD Virus Isolation Following Challenge BVD VirusIsolation Frequency (%) of LSMean Days Treatment Viremic Animals withViremia T01 Saline 10/10 (100.0) 8.4 T02 Amphigen, Quil A 3/10 (30.0)0.3 T03 Amphigen, 2 mg GPI-0100, 0/10 (0.0)  0.0 AIOH T04 Amphigen, 2 mgGPI-0100 1/10 (10.0) 0.1 T05 Amphigen, 3 mg GPI-0100 3/10 (30.0) 0.3 T06Amphigen, 5 mg GPI-0100 2/10 (20.0) 0.2

[0163] TABLE 14 Clinical Signs of BVD Disease Following ChallengeFrequency Frequency (%) Observations with (%) with Clinical DiseaseScore of Total Treatment Disease 0 1 2 Obs. T01 Saline  5/10 (50.0) 103(60.6) 55 (32.4)  12 (7.1) 170 T02 Amphigen, Quil A  5/10 (50.0) 115(67.6) 48 (28.2)   7 (4.1) 170 T03 Amphigen, 2 mg 0/10 (0.0) 128 (75.3)42 (24.7) 0 (0) 170 GPI-0100, AlOH T04 Amphigen, 2 mg 0/10 (0.0) 124(72.9) 46 (27.1) 0 (0) 170 GPI-0100 T05 Amphigen, 3 mg 0/10 (0.0) 104(61.2) 66 (38.8) 0 (0) 170 GPI-0100 T06 Amphigen, 5 mg 0/10 (0.0) 128(75.3) 42 (24.7) 0 (0) 170 GPI-0100

[0164] TABLE 15 Leukopenia Following Challenge Leukopenia Frequency (%)of LSMean Days with Treatment Leukopenic Animals Leukopenia T01 Saline9/10 (90.0) 8.7 T02 Quil A 6/10 (60.0) 1.6 T03 2 mg GPI-0100, AIOH 7/10(70.0) 2.6 T04 2 mg GPI-0100 4/10 (40.0) 1.5 T05 3 mg GPI-0100 7/10(70.0) 2.6 T06 5 mg GPI-0100 8/10 (80.0) 2.9

[0165] In conclusion, safety of each vaccine was demonstrated by theabsence of adverse reactions or mortality in the vaccinated animals.Potency of each vaccine was demonstrated by seroconversion (SVN antibodytiters to BVD-1 and BVD-2>1:8) in 100% of the vaccinated animals.Satisfactory resistance to challenge was demonstrated by the vaccineadjuvanted with 2 mg GPI-0100 only.

EXAMPLE 16 Vaccine Preparation Containing Microencapsulated Antigen inMicrofluidized Oil-In-Water Emulsion

[0166] Three grams of Trehalose (Fluka) was added to water to get astock of 333mg/ml of Trehalose solution. Recombinant PauA antigensolubililzed in 0.8% SDS solution (SDS/rPauA) was added to Trehalosesolution to get a final concentration of 494 μg rPauA/ml. In the nextstep 10 grams of polylactide glycolic acid (PLG- Resomer RE 503H,Boeringher Ingelheim) was dissolved in 200 ml Methylene Chloride(MeCl2). The resulting PLG/MeCl2 solution was combined with theSDS-rPauA/trehalose solution prepared in the first step. The combinedsolution was subjected to microfluidization using (Microfluidizer fromMicrofluidics Model M110EH) and the microfluidized preparation was spraydried using (Temco Spray Dryer Model SD-05). The spray dried materialwas collected using a 500 micron screen.

[0167] The concentration of rPauA in this spray dried material wasquantified using a Western blot analysis. 1.04 mg of spray-driedmaterial was dissolved in 50 μl of acetone and centrifuged at 13,200 rpmat room temperature for 10 minutes. The supernatant was removed. Thesupernatantand the pellet fractions were dried in a biological safetyhood for 2.5 hours. The pellet was resuspended in 47.43 μL of samplesolution (25 μl of sample buffer +10 μl of reducing agent+65 μl ofwater). The dried supernatant fraction was resuspended with 20 μl ofsample solution. In the western analysis purified PauA was used as astandard to quantify the rPauA content of the spray dried material.

[0168] A 20% Manitol stock solution was prepared by dissolving 100 gramsof mannitol (Sigma) in 500 ml of Water for Injection (WFI). Solution washeated to 40° C. with hot plate/stirrer and cooled to 30° C. Solutionwas sterile filtered through a 0.22 micron sterile filter (Millipore).2.5% Carboxymethylcellulose solution was prepared by dissolving 12.5grams of carboxymethyulcellulose (Sigma) in 500 ml of WFI and mixedovernight at 4° C. Solution was autoclaved at 121° C.

[0169] The powder resulting from spray drying was reconstituted in asolution containing 5% mannitol, 0.3% carboxymethyl cellulose, and1:5000 of thimerosol. The final solution was aliquoted in to 3 ml vialsand lyophilized using a Lyophilizer (USIFROID). The lyophilized powderrepresents the microencapsulated rPauA. The microencapsulated subunitprotein antigen is resuspended in 2 ml of microfluidized oil-in-wateremulsion containing an AMPHIGEN® formulation (such as the microfluidizedemulsion described in Example 20) and used as a vaccine.

EXAMPLE 17 Preparation of Microfluidized Vaccine Formulation ContainingBoth Bacterial Whole Cell Antigen and Recombinant Protein Antigen inOil-In-Water Emulsion

[0170] Two vaccine preparations were made which contained bothrecombinant Streptococcus uberis PauA protein and Escherichia colibacterial cells, added intrinsically to oil-in-water emulsions asdescribed in Examples 2 and 3. The recombinant PauA antigen was at theconcentration of 100 μg per dose and the E. coli cells were at the finalcount of 4×10 per dose. The emulsion adjuvant compositions of the twovaccine formulations are shown in the Table 16. TABLE 16 Vaccineformulations containing both the recombinant protein and whole E. colicells. Treatment Antigen Adjuvant T01 Placebo Saline T02 Pau A/E. coliSEAM-14 T03 Pau A/E. coli 2.5% Amphigen, 0.5 mg GPI-0100, 0.5 mgcholesterol T04 Pau A/E. coli 2.5% Amphigen, 0.5 mgdimethyldioctadecylammonium bromide (DDA), 0.5 mg cholesterol

EXAMPLE 18 Immune Response to Microfluidized Vaccine Containing therPauA and Whole Cell Bacterial Agents in Oil-In-Water Emulsion

[0171] Mature dairy cows were used in this experiment. Animals were atthe end of their first or second lactation at the time of enrollment.Two ml of each vaccine formulation was administered subcutaneously threetimes, once at the time of drying off (D-0), 28 days later (D=28), andagain 4 to 10 days following calving (C+4 - C+10). The first and thirddose was administered on the left side of the neck and the second dosewas administered on the right side of the neck. Blood was collectedprior to each vaccination and approximately 14 days and 32 daysfollowing third vaccination. The antibody titer for E. coli and therPauA antigen were determined through ELISA. As shown in FIG. 8, theresults indicate that the antibody titer for rPauA was higher in thegroup vaccinated with vaccine formulation containing GPI-0100 as animmunostimulant and peaked on day 70 post initial vaccination. Theantibody titer for E. coli antigen is shown in FIG. 9. The antibodytiter for E. coli antigen was comparable in both vaccine formulations,although the presence of GPI-0100 as an immunostimulant induced arelatively higher antibody titer when compared to the formulation withDDA as an immunostimulant.

EXAMPLE 19 Analysis of Virucidal Activity of the MicrofluidizedAMPHIGEN® Formulation Based Vaccine Preparations

[0172] In order to determine whether microfluidization inactivates thevirus, the viricidal activity of three microfluidized AMPHIGEN®formulation based vaccine preparations were determined. The threepreparations contained three different bovine infectious viruses, namelybovine herpes virus (BHV), parainfluenza virus 3 (PI3), and bovinerespiratory synctial virus (BRSV).

[0173] Detection of the viricidal activity in the three vaccinepreparations was conducted in accordance with the USDA 9CFR.113.35requirements.

[0174] The results shown in Table 16 indicate that microfluidization ofAMPHIGEN® formulation-based vaccine preparations does not cause anysignificant inactivation of the vaccine preparation. TABLE 16 AnalysisOf Viricidal Activities Of Microfluidized Vaccines Serial BRSV BHV PI3 A0 0.2 0 AM200 −0.2 0 −0.2 AM75 0 −0.3 −0.3 AM75@37 C. 0.1 −0.3 −0.2 B 0−0.1 −0.2 BM200 0 0 −0.2 BM75 −0.2 −0.5 0 BM75@37 C. 0.5 −0.5 0 C 0.1−0.1 −0.2 CM200 −0.2 −0.1 −0.2 CM75 0.1 0.5 −0.2 CM75@37 C. 0.5 0.5 −0.2

[0175] A value above 0.7 is an indication of viricidal effect.

EXAMPLE 20 Preparation of a Microfluidized AMPHIGEN® Formulation

[0176] An AMPHIGEN® formulation was prepared by combining the DRAKEOLlecithin oil solution (light mineral oil with 25% lecithin) and TWEEN 80(with the final concentration of 0.18%) and Span 80 (with the finalconcentration of 0.08%) with mixing for 8-22 hours at 36±1° C. The oilmixture was then added to saline with the aide of a Ross® (Hauppauge,N.Y. 11788) emulsifier at approximately 3400 rpm. Subsequently themixture was passed once through a microfluidizer with a 200 μminteraction chamber at 4500±500 psi. FIGS. 10A and 10B show thestability of the microfluidized AMPHIGEN® formulation. Particle sizedistribution, as measured by laser diffraction, at the starting, initialtime point (FIG. 10A) was nearly identical to the particle sizedistribution after 22 months of 4° C. storage (FIG. 10B).

What is claimed is:
 1. A submicron oil-in-water emulsion useful as avaccine adjuvant comprising a light hydrocarbon non-metabolizable oil, asurfactant, and an aqueous component, wherein said oil is dispersed insaid aqueous component and the mean oil droplet size is less than 1 μm.2. The emulsion of claim 1, wherein said oil is in an amount of 1% to50% v/v, and said surfactant is in an amount of 0.01% to 10% v/v.
 3. Theemulsion of claim 1 wherein said mean droplet size is less than 0.8 μm.4. The emulsion of claim 3 wherein said mean droplet size is between 0.1to 0.5 μm.
 5. The emulsion of claim 1 wherein said light hydrocarbonnon-metabolizable oil is light mineral oil.
 6. The emulsion of claim 1wherein said surfactant comprises a phospholipid compound or a mixtureof phospholipid compounds.
 7. The emulsion of claim 6 wherein saidphospholipid is selected from the group consisting ofphosphstidylchloine, phosphatidylethanolmine, phosphatidylserine,phosphatidylethanolmine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, phosphatidic acid, spingomyelin and cardiolipin.8. The emulsion of claim 6 wherein said mixture of phospholipidcompounds is lecithin.
 9. The emulsion of claim 1, wherein saidsurfactant comprises at least one of TWEEN or SPAN.
 10. A submicronoil-in-water emulsion useful as a vaccine adjuvant comprising about 40%v/v of mineral oil, about 10% w/v of lecithin, about 0.18% v/v ofTWEEN®-80, about 0.08% v/v of SPAN®-80, and an aqueous phase, whereinsaid oil is dispersed in said aqueous phase and the mean oil dropletsize is between 0.1 μm to 0.5 μm.
 11. The emulsion of claim 1 or 10,further comprising an immunostimulatory molecule selected from Quil-A,GP-100, cholesterol or DDA.
 12. A method of preparing a submicronoil-in-water emulsion, comprising: (a) preparing a mixture by combininga light hydrocarbon non-metabolizable oil, a surfactant, and an aqueouscomponent; (b) subjecting said mixture to a primary emulsificationprocess to produce an oil-in-water emulsion which has a mean oil dropletsize of 1.0 μm to 1.1 μm ; and (c) subjecting the oil-in-water emulsionprepared in (b) to microfluidization to produce said submicronoil-in-water emulsion, wherein the submicron emulsion has a mean oildroplet size of less than 1 μm.
 13. The method of claim 12, wherein saidoil is in an amount of 1% to 50% v/v, and said surfactant is in anamount of 0.01% to 10% v/v.
 14. The method of claim 13 wherein said meanoil droplet size in said submicron oil-in-water emulsion is less than0.8 μm.
 15. The method of claim 14 wherein said mean oil droplet size insaid submicron oil-in-water emulsion is between 0.1 -0.5 μm.
 16. Themethod of claim 12 wherein said light hydrocarbon non-metabolizable oilis mineral oil.
 17. The method of claim 12 wherein said surfactantcomprises a phospholipid compound or a mixture of phospholipidcompounds.
 18. The method of claim 17 wherein said phospholipid isselected from the group consisting of phosphstidylchloine,phosphatidylethanolmine, phosphatidylserine, phosphatidylethanolmine,phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,phosphatidic acid, spingomyelin and cardiolipin.
 19. The method of claim17 wherein said mixture of phospholipid compounds is lecithin.
 20. Themethod of claim 12 wherein said surfactant comprises at least one ofTWEEN® or SPAN®.
 21. The method of claim 12 wherein saidmicrofluidization is conducted in a microfluidizer at an operatingpressure in the range of about 1,000 to 15,000 psi.
 22. The method ofclaim 12, wherein the mixture formed in step (a) further includes animmunostimulatory molecule selected from Quil-A, GP-100, cholesterol orDDA.
 23. A submicron oil-in-water emulsion prepared according to any oneof the methods of claims 12-22.
 24. A vaccine composition comprising anoil-in-water emulsion and an antigen, wherein said antigen is dispersedin said emulsion, said emulsion comprises a light hydrocarbonnon-metabolizable oil, a surfactant and an aqueous component, andwherein the mean oil droplet size of said emulsion is less than 1 μm.25. The vaccine composition of claim 24 wherein said oil is present insaid vaccine composition in an amount of 1% to 20% v/v, and saidsurfactant is present in said vaccine composition in an amount of 0.01%to 10% v/v.
 26. The vaccine composition of claim 24 wherein said meandroplet size is in the range of less than 0.8 μm.
 27. The vaccinecomposition of claim 26 wherein said mean droplet size is between 0.1 to0.5 μm.
 28. The vaccine composition of 24 wherein said light hydrocarbonnon-metabolizable oil is light mineral oil.
 29. The vaccine compositionof claim 24 wherein said surfactant comprises a phospholipid compound ora mixture of phospholipid compounds.
 30. The vaccine composition ofclaim 29 wherein said phospholipid is selected from the group consistingof phosphstidylchloine, phosphatidylethanolmine, phosphatidylserine,phosphatidylethanolmine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, phosphatidic acid, spingomyelin and cardiolipin.31. The vaccine composition of claim 29 wherein said mixture ofphospholipid compounds is lecithin.
 32. The vaccine composition of claim24, wherein said surfactant comprises at least one of TWEEN® or SPAN®.33. The vaccine composition of claim 24, further comprising animmunostimulatory molecule selected from Quil-A, GP-100, cholesterol orDDA.
 34. The vaccine composition of claim 24, wherein said antigencomprises a viral antigen.
 35. The vaccine composition of claim 34,wherein said viral antigen comprises killed Bovine Viral Diarrhea virusType 1 or Type
 2. 36. The vaccine composition of claim 24, wherein saidantigen comprises a bacterial antigen.
 37. The vaccine composition ofclaim 36, wherein said bacterial antigen comprises at least one of aninactivated Leptospira bacterin, the recombinant Streptococcus uberisPauA protein, or an E. coli cell preparation.
 38. A method of preparinga vaccine composition, comprising: (a) preparing a mixture by combininga light hydrocarbon non-metabolizable oil, a surfactant, and an aqueouscomponent; (b) combining an antigen with the mixture formed in (a); (c)subjecting the mixture containing said antigen, which is formed in (b),to a primary emulsification process to produce an oil-in-water emulsionwhich has a mean oil droplet size of 1.0 μm to 1.1 μm; and (d)subjecting the emulsion formed in (c) to high pressrure homogenizationto produce said vaccine composition, wherein the composition has a meanoil droplet size of less than 1 μm.
 39. The method of claim 38, whereinthe antigen to be combined with the mixture formed in (a) is provided ina mixture comprising a saponin and a sterol that is formed by: (i)combining said antigen with said saponin to form a mixture; (ii)subjecting the mixture formed in (i) to homogenization; (iii) addingsaid sterol to the homogenized mixture formed in (ii); and (iv)subjecting the mixture formed in (iii) to homogenization.
 40. A methodof preparing a vaccine composition, comprising: (a) combining an antigenwith a saponin to form a mixture; (b) subjecting the mixture formed in(a) to homogenization; (c) adding a sterol to the homogenized mixtureformed in (b); (d) subjecting the mixture formed in (c) tohomogenization; (e) preparing a mixture of a light hydrocarbonnon-metabolizable oil, a surfactant, and an aqueous component; (f)adding the mixture of (e) to the homogenized mixture formed in (d); (g)subjecting the mixture formed in (f) to further homogenization toproduce an oil-in-water emulsion which has a mean oil droplet size of1.0 μm to 1.1 μm; and (h) subjecting the emulsion formed in (c) to highpressure homogenization to produce said vaccine composition, wherein thecomposition has a mean oil droplet size of less than 1 μm.
 41. Themethod of claim 38 or 40, wherein said oil is present in the vaccinecomposition in an amount of 1% to 20% v/v, and said surfactant ispresent in said vaccine composition in an amount of 0.01% to 10% v/v.42. The method of claim 38 or 40 wherein said mean oil droplet size insaid vaccine is less than 0.8 μm.
 43. The method of claim 42 whereinsaid mean oil droplet size is between 0.1 to 0.8 μm.
 44. The method ofclaim 38 or 40 wherein said light hydrocarbon non-metabolizable oil islight mineral oil.
 45. The method of claim 38 or 40 wherein saidsurfactant comprises a phospholipid compound or a mixture ofphospholipid compounds.
 46. The method of claim 45 wherein saidphospholipid is selected from the group consisting ofphosphstidylchloine, phosphatidylethanolmine, phosphatidylserine,phosphatidylethanolmine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, phosphatidic acid, spingomyelin and cardiolipin.47. The method of claim 45 wherein said mixture of phospholipidcompounds is lecithin.
 48. The method of claim 38 or 40 wherein saidsurfactant comprises at least one of TWEEN or SPAN.
 49. The method ofclaim 39 or 40, wherein said saponin is Quil A and said sterol ischolesterol.
 50. The method of claim 38 or 40 wherein said high pressurehomogenization is conducted in a microfluidizer at an operating pressurein the range of about 1,000 to 15,000 psi.
 51. The method of claim 38 or40, wherein said antigen comprises a viral antigen.
 52. The method ofclaim 51, wherein said viral antigen comprises killed Bovine ViralDiarrhea virus Type 1 or Type
 2. 53. The method of claim 38 or 40,wherein said antigen comprises a bacterial antigen.
 54. The method ofclaim 53, wherein said bacterial antigen comprises at least one of aninactivated Leptospira bacterin, the recombinant Streptococcus uberisPauA protein, or an E. coli cell preparation.
 55. A vaccine preparedaccording to any one of the methods of claims 38-40.
 56. A vaccinecomposition comprising an microencapsulated antigen and an oil-in-wateremulsion, wherein said microencapsulated antigen is dispersed in saidemulsion, and said emulsion comprises a light hydrocarbonnon-metabolizable oil, a surfactant and an aqueous component, andwherein the mean oil droplet size of said emulsion is less than 1 μm.57. The vaccine composition of claim 56 wherein said oil is present insaid vaccine composition in an amount of 1.0% to 20% v/v, and saidsurfactant is present in said vaccine composition in an amount of 0.01%to 10% v/v.
 58. The vaccine composition of claim 56 wherein said meandroplet size is in the range of less than 0.8 μm.
 59. The vaccinecomposition of claim 58 wherein said mean droplet size is between 0.1 to0.5 μm.
 60. The vaccine composition of 56 wherein said light hydrocarbonnon-metabolizable oil is light mineral oil.
 61. The vaccine compositionof claim 56 wherein said surfactant comprises a phospholipid compound ora mixture of phospholipid compounds.
 62. The vaccine composition ofclaim 61 wherein said mixture of phospholipid compounds is lecithin. 63.The vaccine composition of claim 56, wherein said surfactant comprisesat least one of TWEEN or SPAN.
 64. The vaccine composition of claim 56,further comprising an immunostimulatory molecule selected from Quil-A,GP-100, cholesterol or DDA.
 65. The vaccine composition of claim 56,wherein said antigen is a viral antigen or a bacterial antigen.
 66. Thevaccine composition of claim 56, wherein said antigen is encapsulated ina particulate carrier, and wherein said carrier comprises polylactideglycolic acid.